Halbig, D. (Corresponding author)

1999
Forschungszentrum, Zentralbibliothek Jülich

Report No.: Juel-3661

Abstract: The periplasmic NADP-containing glucose-fructose oxidoreductase (GFOR) of the gramnegative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twinarginine motif. The replacement of one or both arginine residues results in drastically reduced translocation of GFOR to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type GFOR, bind NADP in a looser and dissociable manner were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of GFOR into the periplasm uses a Sec-independent apparatus which recognizes, as additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore these results suggest that GFOR is exported in a folded form. A GFOR::~-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of cytosolic preform of wild-type GFOR expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the GFOR translocation apparatus can be blocked without interfering with the export of essential proteins via the Secpathway. In higher plant chloroplasts, twin-arginine signal peptides are specific targeting signals for the Sec-independent ilpH-pathway of the thylakoid membrane system. In agreement with the assumed phylogenetic origin of this protein transport mechanism, GFOR can be efficiently translocated by the ilpH-dependent pathway when analyzed with isolated thylakoid membranes. Transport is sensitive to the ionophore nigericin and competes with specific substrates for the Aplf-dependent transport route. In contrast, neither sodium azide nor enzymatic destruction of the nucleoside triphosphates in the assays affects thylakoid transport of GFOR indicating that the Sec-apparatus is not involved in this process. Mutagenesis of the twin-arginine motif in the GFOR signal peptide prevents membrane translocation of the protein emphasizing the importance of these residues for the transport process

Keyword(s): membrane transport ; biological membrane ; bacterium ; protein secretion ; bioengineering ; reductase

1. Institut für Biotechnologie (IBT)